Na-H exchanger NHE3 is responsible directly and indirectly for the majority of NaHCO3 and NaCl reabsorption in the proximal tubule. Phosphorylation sites mediating NHE3 inhibition in response to PKA (S552 and S605), and stimulation in response to SGK1 (S661) and CK2 (S716) have been described. We have recently identified an additional novel phosphorylation site (S791) that stimulates NHE3 activity. The general goal of this project is to elucidate how combinatorial patterns of NHE3 phosphorylation integrate to alter NHE3 trafficking and activity. To accomplish this goal, we will pursue the following specific aims: Aim 1. Generate and confirm specificity of new phosphospecific polyclonal and monoclonal anti-NHE3 antibodies raised against S661, S716, and S791 containing phosphopeptides. Together with our previously generated antibodies to the two PKA sites, these new antibodies will provide us with a unique set of reagents to understand the integrated regulation of NHE3 by phosphorylation in response to physiologic stimuli that regulate proximal tubule transport. Aim 2. Use this panel of phosphospecific antibodies to evaluate the combinatorial patterns of phosphorylation of endogenous NHE3 at multiple residues in native rat kidney in vivo at baseline, and in response to stimuli that either inhibit (e.g. dopamine, PTH) or enhance (e.g. dexamethasone, angiotensin II) NHE3 activity. Compare subcellular localization of phosphorylated NHE3 and total NHE3 in response to these stimuli by immunoblotting of subcellular membrane fractions and by immunofluorescence microscopy. The data from these studies will allow us to generate mechanistic hypotheses concerning the roles of phosphorylation at specific sites or combinations of sites in governing activity and trafficking of NHE3 in vivo. Aim 3. Use OKP proximal tubule cells as a model system to refine and test the hypotheses generated in the preceding aim concerning the roles of phosphorylation at specific sites or combinations of sites in regulating NHE3. Verify changes in patterns of phosphorylation of endogenous NHE3 in OKP cells in response to those stimuli found to alter phosphorylation of NHE3 in vivo. Evaluate temporal sequence of phosphorylation at different sites in NHE3. Compare patterns of phosphorylation of surface and intracellular NHE3 by use of biotinylation studies. Correlate the kinetics of phosphorylation changes with changes in transporter surface expression and activity. Perform mutagenesis studies using transfected NHE3 to test the roles of phosphorylation at specific sites or combinations of sites in governing NHE3 intrinsic activity and trafficking. Aim 4. Identify downstream effector proteins that associate with NHE3 phosphorylated at specific sites or combinations of sites and that mediate changes in NHE3 intrinsic activity and trafficking. Perform co-precipitation studies with antibodies against known NHE3- associated proteins (e.g. megalin, NHERF1, ezrin, PP2A) to determine possible selectivity for NHE3 phosphorylated at specific combinations of residues. Use affinity chromatography and mass spectrometry to identify novel proteins that associate with NHE3 phosphorylated at specific sites or combinations of sites.